1. Field of the Invention
The present invention relates to isolated cDNA sequences coding for murine interleukin 5 receptors, murine secretory interleukin 5 receptors and human interleukin 5 recepters and to murine interleukin 5 receptors, murine secretory interleukin 5 receptors and human interleukin 5 receptors which are produced using the isolated cDNA sequences as well as to methods of producing the interleukin 5 receptors.
2. Prior Art
Interleukin 5 (referred to as "IL-5", hereinafter) is a proliferation and differentiation factor for eosinophils and B lineage cells (Immunol. Rev. 102: 29, 107.,1988). It has been known that IL-5 is produced especially by T cells primed with Mycobacterium tuberculosis, parasites or allo-antigens (J. Immunol. 140: 1175, 1988; Nature, 324: 70, 1986). IL-5 has also been known to induce production of IgM class immunoglobulin including anti-DNA antibody. Recently, IL-5 has been suspected of involvement in autoimmune diseases and there is a report that IL-5 is closely associated with eosinophilia accompanied by autoantibody production, fascitis and myositis (Eosinophils, Oxford University Press, 1988).
There are two types of IL-5 receptors (referred to as "IL-5R", hereinafter), namely, membrane bound IL-5R and secretory IL-5R. Among them, mouse secretory IL-5R is able to bind to human IL-5 and therefore expected to serve as a therapeutic agent for diseases associated with IL-5.
The inventors have obtained IL-5 responsive early B cells, T 88 and T-88M by culturing mouse bone marrow cells in the presence of IL-5 (Growth Factors 1: 135 1989) and produced IL-5R. The cross-linking reaction and subsequent SDS-PAGE analysis have revealed that IL-5R comprises at least two types of subunits, one having a molecular weight of about 46,500 and the other having a molecular weight of about 114,000, and that there are two types of IL-5R, a low affinity IL-5R having the dissociation constant of 27 nM and a high affinity IL-5R having the dissociation constant of 150 pM. It has been believed that the low affinity IL-5R comprises the small subunit of an estimated molecular weight of 46,500 while a high affinity IL-5R comprises the large subunit (MW: 114,000) and the small subunit (46,500) (Proc. Natl. Acad. Sci. USA 86: 2311, 1989).
The inventors have produced H7 and T21 monoclonal antibodies by immunizing rats with a membrane fraction of T88-M cells, which inhibit the binding of IL-5 to IL-5R (Int. Immunol. 2: 181, 1990; J. Immunol. 144: 4218, 1990). Anti-IL-5R antibodies, H7 and T21, are found to bind to glycoprotein of the molecular weight of about 60,000 according to the SDS-PAGE analysis. The real molecular weight of the small subunit is found to be about 55,000 according to the binding assay using IL-5 free of an oligosaccharide, suggesting that the low affinity IL-5R comprises a single molecule of molecular weight of about 60,000 (Int. Immunol. 2: 181, 1990).
We have also reported recently that IL-5R is found on the cell surface of human eosinophils. The dissociation constant of human IL-5R is 170-330 pM and the molecular weight is 55,000-60,000 according to the SDS-PAGE analysis. Human IL-5R appeares to be comparable to a low affinity murine IL-5R (Migita, M., Yamaguchi, N., Mita, S., Higuchi, S., Hitoshi, Y., Yoshida, Y., Tomonaga, M., Matsuda, I., Tominaga, A., Takatsu, K., 1991, Cellular Immunology, 133: 484-497).
There has been no report on the isolation of a DNA sequence coding for the low affinity murine/human IL-5R. An object of the invention is to isolate the DNA sequence coding for the low affinity murine/human IL-5R and to determine the DNA sequence. The isolated DNA sequence may be used to produce murine/human IL-5R in mammalian cells. Another object of the invention is to obtain a DNA sequence coding for secretory IL-5R which is distinct from the DNA sequence coding for membrane bound IL-5R and to produce pure secretory IL-5R using the DNA.
The present invention is characterized by the following description:
(1). An isolated cDNA sequence coding for murine interleukin 5 receptor which is synthesized from murine early B cell mRNA.
(2). The isolated cDNA sequence of (1) wherein the nucleotide sequence comprises the open reading frame sequence described in SEQ ID No. 1.
(3) The isolated cDNA sequence of (1) wherein the nucleotide sequence comprises the entire sequence described in SEQ ID No. 2.
(4). An isolated cDNA sequence coding for secretory murine IL-5R which is synthesized from murine early B cell mRNA.
(5) The isolated cDNA sequence of (4) wherein the nucleotide sequence comprises the open reading frame sequence described in SEQ ID No. 3.
(6). The isolated cDNA sequence of (4) wherein the nucleotide sequence comprises the entire sequence described in SEQ ID No. 4.
(7). An isolated murine interleukin 5 receptor wherein the amino acid sequence comprises the sequence described in SEQ ID No. 5.
(8). An isolated murine interleukin 5 receptor wherein the amino acid sequence comprises the sequence described in SEQ ID No. 6.
(9) An isolated murine secretory interleukin 5 receptor wherein the amino acid sequence comprises the sequence described in SEQ ID No. 7.
(10) An isolated murine secretory interleukin 5 receptor wherein the amino acid sequence comprises the sequence described in SEQ ID No. 8.
(11). A method of producing the murine interleukin 5 receptors which comprises culturing cells capable of expressing the murine interleukin 5 receptors in medium and isolating the murine interleukin 5 receptors from the cells or the culture supernatant using anti-interleukin 5 receptor antibodies.
(12) A COS 7 monkey cell (ATCC CRL 1651) transfected with a recombinant vector containing the cDNA sequence of any one of (1)-(6).
(13) A method of producing the murine interleukin 5 receptors and the murine secretory interleukin 5 receptors comprises culturing the COS 7 cell transfected with relevant DNA in medium, and recovering the murine interleukin 5 receptors from the cells or secretory murine interleukin 5 receptors from the culture supernatant.
(14). An isolated cDNA sequence coding for human interleukin 5 receptor which is synthesized from mRNA of a human peripheral blood eosinophil.
(15). The isolated cDNA sequence of (14) wherein the nucleotide sequence described in SEQ ID No. 9 comprises the open reading frame sequence coding for human interleukin 5 receptor.
(16). The isolated cDNA sequence of (14) wherein the nucleotide sequence described in SEQ ID No. 10 comprises the entire sequence coding for human interleukin 5 receptor.
(17). The isolated cDNA sequence of (14) wherein the nucleotide sequence described in SEQ ID No. 11 comprises the open reading frame sequence coding for human interleukin 5 receptor 2.
(18). The isolated cDNA sequence of (14) wherein the nucleotide sequence described in SEQ ID No. 12. comprises the entire sequence coding for human interleukin 5 receptor 2.
(19). An isolated human interleukin 5 receptor wherein the amino acid sequence comprises the sequence described in SEQ ID NO. 13.
(20). An isolated human interleukin 5 receptor wherein the amino acid sequence comprises the sequence described in SEQ ID NO. 14.
(21 ). The isolated cDNA sequence of (14) coding for a whole or part of amino acid residue numbers 1-333 described in SEQ ID No. 13
(22). A secretory human interleukin 5 receptor which lacks a cytoplasmic region and a transmembrane region of human interleukin 5 receptor.
(23). An expression vector comprising the cDNA sequence of any one of (14), (15), (16), (17), (18), and (21).
(24). A method of producing the secretory human interleukin 5 receptor and its analogues which comprises culturing a recombinant vector coding for the secretory human interleukin 5 receptor under the conditions which promote the expression thereof and recovering the secretory human interleukin 5 receptor.